PROJECT SUMMARY/ABSTRACT Cholesterol is an important metabolic precursor, regulator, and component of cellular membranes. Exogenous cholesterol is acquired principally through receptor-mediated endocytosis of cholesteryl ester-laden low-density lipoproteins (LDL) and trafficked to the lysosome where it is distributed to other cellular organelles. While it is well established that bulk export of lysosomal cholesterol is dependent on lysosomal protein NPC1, NPC1- dependent and lower flux, NPC1-independent post-lysosomal cholesterol trafficking pathways are poorly understood. This is, in part, due to a lack of suitable cholesterol trafficking probes. Diazirine-alkyne (DA) functionalized probes are structurally minimally modified and enable tagging and enrichment of protein targets for identification by mass spectrometry. DA-probes provide a powerful new tool for the discovery and study of previously elusive post-lysosomal cholesterol trafficking pathways. I hypothesize the specific itinerary and metabolic fate of lipoprotein-derived cholesterol involves multiple, distinct distribution pathways and is mediated by proteins associated with the limiting lysosomal membrane and non-lysosomal trafficking proteins associated with diverse subcellular compartments. In Specific Aim 1 I will characterize novel DA-cholesterol probes that I have developed as tools for the study of cholesterol trafficking. In Specific Aim 2 I will use validated DA-probes to identify proteins involved in the post-lysosomal cholesterol trafficking network. To specifically assess post- lysosomal trafficking, DA-probe will be delivered in reconstituted LDL. Knock-out analysis of key trafficking nodes, coupled with biochemical trafficking assays will position identified proteins in specific trafficking pathways. In Specific Aim 3 I will identify lower flux, NPC1-independent post-lysosomal cholesterol trafficking pathways in NPC1-deficient cells using the labeling approach described. A proteomic strategy combined with biochemical assays will be used to determine the fate of released cholesterol. The long-term objective of this project is to elucidate the major nodes through which lipoprotein-derived cholesterol trafficks from the lysosome. This goal aligns well with the mission of NHLBI and may lead to discovery of novel targets for treating both genetic (e.g., NPC disease) and acquired (e.g., atherosclerosis) cholesterol storage disorders.